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Kit for diagnosing or detecting leukemia

A kit and inhibitor technology, which is applied in the field of real-time fluorescence quantitative RT-PCR detection kits, can solve the problems of insufficient sensitivity and inaccurate quantification, and achieve the effects of accurate quantification, good repeatability and convenient detection.

Inactive Publication Date: 2012-07-04
ARMY MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Genetic diagnosis plays an important role in the diagnosis of leukemia, observation of curative effect, and judgment of prognosis. The methods for genetic diagnosis of leukemia mainly include fluorescence in situ hybridization (FISH) technology and conventional RT-PCR technology, but these two methods are not enough. Sensitive, quantitative inaccurate

Method used

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  • Kit for diagnosing or detecting leukemia
  • Kit for diagnosing or detecting leukemia
  • Kit for diagnosing or detecting leukemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of real-time fluorescent quantitative RT-PCR detection kit for Wnt5a gene mRNA

[0030] This kit includes the following components:

[0031] a. Lymphocyte separation medium, Trizol total RNA extraction solution, 5× reverse transcription buffer, 200U / μL M-MLV reverse transcriptase, 500ng / μL reverse transcription primer Oligo(dT) 18 , 40U / μL RNase inhibitor, DEPC-H 2 O, 10×quantitative PCR buffer, 10U / μL Taq DNA polymerase, 10mM dNTPs solution. (lymphocyte separation liquid was purchased from Tianjin TBD Company, and Trizol total RNA extraction liquid was purchased from Invitrogen Company)

[0032] b. Wnt5a gene standard product: recombinant pMD-19 plasmid containing Wnt5a partial coding sequence, its concentration is 10 8 copy / μL, the Wnt5a nucleotide sequence contained is:

[0033] 5'-AGTTCTTCCTAGTGGCTTTGGCCATATTTTTCTCCTTCGCCCAGGTTGTAATTGAAGCCAATTCTTGGTGGTCGCTAGGTATGAATAACCCTGTTCAGATGTCAGAAGTATATATTATAGGAGCAGCCAACTGGCAGGACTTTCTCAAGGACAGAAGAAA...

Embodiment 2

[0053] Example 2 Detection of Wnt5a gene mRNA real-time fluorescent quantitative RT-PCR detection kit

[0054] A positive control and a negative control are set up for each detection of this kit, and its detection method includes the following steps:

[0055] ① Separation of nucleated cells from bone marrow or peripheral blood: add fresh anticoagulated bone marrow to an equal volume of normal saline to dilute, and mix well; After dilution, the bone marrow was carefully added to the surface of the separation liquid to keep a clear interface, and centrifuged at 2000rpm for 15min; carefully aspirate the nucleated cell layer into a centrifuge tube, washed twice with normal saline, and centrifuged at 500rpm for 5min each time.

[0056] ②Extraction of total RNA: add 1.0mL Trizol to the isolated cells, mix thoroughly, and let stand at room temperature for 5 minutes; add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 2-3 minutes, and centrif...

Embodiment 3

[0061] Example 3 Kit performance evaluation:

[0062] In order to detect the repeatability and sensitivity of the Wnt5a gene mRNA real-time fluorescent quantitative PCR method, the present invention uses repeatability experiments and sensitivity experiments for evaluation.

[0063] 1. Standard curve preparation and linear range

[0064] Get the Wnt5a and GAPDH gene plasmid standards (10 8 -10 1 copies / μL) for fluorescent quantitative PCR reaction. Reaction conditions: pre-denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 10 seconds, annealing at 58°C, and extension for 45 seconds, a total of 40 cycles, and fluorescence signals were collected at the annealing temperature. According to the fluorescent signal detected by the instrument, it is processed by software (Bio-rad company iQ5 TM Quantitative PCR instrument comes with) to obtain the standard curve, and calculate the correlation coefficient (R), get the linear range, see Figure 1a , 1b and ...

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Abstract

The invention relates to a kit for diagnosing or detecting leukemia, which contains a lymphocytes separation medium, total RNA extracting solution, reverse transcription buffer solution, M-MLV reverse transcriptase, a reverse transcription primer Oligo(dT)18, an RNA enzyme inhibitor, DEPC water, quantitative PCR buffer solution, Taq DNA polymerase, dNTPs solution, a Wnt5a gene reference standard, an internal reference GAPDH gene reference standard, a primer and a fluorescent probe for detecting a Wnt5a gene and a primer and a fluorescent probe for detecting an internal reference GAPDH gene. The kit for diagnosing or detecting the leukemia has the advantages of simple preparation, convenient detection, rapidness, sensitivity, accurate quantification, high repetitiveness and specificity and suitability for clinical application.

Description

technical field [0001] The invention relates to a real-time fluorescent quantitative RT-PCR detection kit, in particular to a kit for diagnosing or detecting leukemia. Background technique [0002] Leukemia is a clonal disease of hematopoietic stem cells and a group of highly heterogeneous malignant blood diseases. The occurrence of leukemia is related to environmental pollution, increased pressure and other factors, but the root cause is the abnormal expression of genes. Genetic diagnosis plays an important role in the diagnosis of leukemia, observation of curative effect, and judgment of prognosis. The methods for genetic diagnosis of leukemia mainly include fluorescence in situ hybridization (FISH) technology and conventional RT-PCR technology, but these two methods are not enough. Sensitive, quantitative inaccurate. Leukemia needs to be detected as early as possible and treated in time. During the treatment of leukemia, many leukemias after complete remission will even...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 司维柯杨宗林李招权潘静赵宸李军
Owner ARMY MEDICAL UNIV
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